Wnt genes comprise a large family of secreted polypeptides that are expressed in spatially and tissue-restricted patterns during vertebrate embryonic development. Mutational analysis in mice has shown the importance of Wnts in controlling diverse developmental processes such as patterning of the body axis, central nervous system and limbs, and the regulation of inductive events during organogenesis. Although many components of the Wnt signalling pathway have been identified, little is known about how Wnts and their cognate Frizzled receptors signal to downstream effector molecules. Here we present evidence that a new member of the low-density lipoprotein (LDL)-receptor-related protein family, LRP6 (ref. 3), is critical for Wnt signalling in mice. Embryos homozygous for an insertion mutation in the LRP6 gene exhibit developmental defects that are a striking composite of those caused by mutations in individual Wnt genes. Furthermore, we show a genetic enhancement of a Wnt mutant phenotype in mice lacking one functional copy of LRP6. Together, our results support a broad role for LRP6 in the transduction of several Wnt signals in mammals.

Shortly after implantation the mouse embryo comprises three tissue layers. The founder tissue of the embryo proper, the epiblast, forms a radially symmetric cup of epithelial cells that grows in close apposition to the extra-embryonic ectoderm and the visceral endoderm. This simple cylindrical structure exhibits a distinct molecular pattern along its proximal-distal axis. The anterior-posterior axis of the embryo is positioned later by coordinated cell movements that rotate the pre-existing proximal-distal axis. The transforming growth factor-beta family member Nodal is known to be required for formation of the anterior-posterior axis. Here we show that signals from the epiblast are responsible for the initiation of proximal-distal polarity. Nodal acts to promote posterior cell fates in the epiblast and to maintain molecular pattern in the adjacent extra-embryonic ectoderm. Both of these functions are independent of Smad2. Moreover, Nodal signals from the epiblast also pattern the visceral endoderm by activating the Smad2-dependent pathway required for specification of anterior identity in overlying epiblast cells. Our experiments show that proximal-distal and subsequent anterior-posterior polarity of the pregastrulation embryo result from reciprocal cell-cell interactions between the epiblast and the two extra-embryonic tissues.

We describe the successful application of a modified gene-trap approach, the secretory trap, to systematically analyze the functions in vivo of large numbers of genes encoding secreted and membrane proteins. Secretory-trap insertions in embryonic stem cells can be transmitted to the germ line of mice with high efficiency and effectively mutate the target gene. Of 60 insertions analyzed in mice, one-third cause recessive lethal phenotypes affecting various stages of embryonic and postnatal development. Thus, secretory-trap mutagenesis can be used for a genome-wide functional analysis of cell signaling pathways that are critical for normal mammalian development and physiology.

Nodal is expressed at the lateral edges of the mouse node, but its function in this "organizer" tissue remains unknown due to the early lethality of Nodal mutant embryos. Here we used a genetic strategy to selectively remove Nodal activity from the node. Embryos lacking Nodal in the node fail to initiate molecular asymmetry in the left lateral plate mesoderm and exhibit multiple left-right patterning defects. Nodal may also act as a short-range signal to establish a functional midline barrier. Our findings confirm that the mouse node is instrumental in initiating left-right axis specification and identify Nodal as the key morphogen regulating this process. Genetic pathways controlling invariant situs of the major internal organ systems have been described in several vertebrate model systems (for review, see Capdevila et al. 2000;Hamada et al. 2002). In all cases, expression of the TGF-␤ signaling molecule, Nodal, is restricted to the left side of the embryonic axis, where its activity is critical for specifying the embryonic left-right (LR) axis. The upstream cellular and molecular mechanisms that direct asymmetric gene expression have been extensively described in chick. However, relatively little is known about how embryonic symmetry is broken, and how the LR signaling cascade that ultimately controls the positioning and morphogenesis of the internal organs is activated in mammalian embryos.The mouse node is a bilaminar structure found at the rostral end of the primitive streak and has equivalent secondary axes inducing properties as Spemann's organizer in Xenopus (for review, see Beddington and Robertson 1999). It consists dorsally of epiblast and ventrally of the most caudal aspect of the notochordal plate. Several recent studies indicate that the mouse node plays an important role in the establishment of LR asymmetry (Nonaka et al. 1998;Okada et al. 1999). Ultrastructural studies have shown that monocilia present on cells of the exposed ventral surface of the node rotate in a counterclockwise direction and have been suggested to generate a net leftward flow of extraembryonic fluid known as the nodal flow (Sulik et al. 1994;Nonaka et al. 1998). Laterality defects documented in several mouse mutants that lack cilia, or whose cilia motility is compromised, have lead to the hypothesis that the nodal flow results in the localized accumulation of morphogen(s) on the left side of the node, thereby initiating the LR signaling cascade . In keeping with this, artificially reversing the direction of nodal flow in cultured embryos is sufficient to reverse situs (Nonaka et al. 2002). Surgical ablation of the node (Davidson et al. 1999) and mutations affecting node formation (Dufort et al. 1998) both lead to embryonic laterality defects, implying that the node functions as a signaling center during LR axis formation. The node gives rise to midline structures such as the notochord and floor plate that act as a midline barrier necessary for maintaining correct laterality (Dufort et al. 1998;King et al. 1998;Meno et al. 199...